Validation of Analytical Procedures for Type C Medicated Feeds

TABLE OF CONTENTS
Page No.
Introduction 4
Discussion 5
1. Specificity 6
2. Linearity 7
3. Range 7
4. Accuracy 8
5. Precision 8
5.1. Repeatability 8
5.2. Intermediate Precision 8
5.3. Reproducibility 9
5.4. Proof of Performance 9
6. Limit of Detection 9
6.1. Based on Visual Evaluation 9
6.2. Based on Signal-to-Noise 9
6.3. Based on the Standard Deviation of the Response and the Slope 10
6.3.1. Based on the Standard Deviation of the Blank 10
6.3.2. Based on the Calibration Curve 10
7. Limit of Quantitation 10
7.1. Based on Visual Evaluation 10
7.2. Based on Signal-to-Noise Approach 11
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7.3. Based on the Standard Deviation of the Response and the Slope 11
7.3.1. Based on the Standard Deviation of the Blank 11
7.3.2. Based on the Calibration Curve 11
8. Robustness / Ruggedness 11
9. System Suitability Testing 12
10. Recommended Data 12
Glossary 15
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Guidance for Industry
Validation of Analytical Procedures for Type C
Medicated Feeds1
INTRODUCTION
The purpose of this guidance is to provide recommendations on how to consider the
various validation characteristics for each analytical procedure used in medicated
feed assays. This guidance is written primarily for chromatographic methods;
however, the guidance does not limit the analytical technique to chromatographic
procedures, as other techniques may be appropriate. In some cases (for example,
demonstration of specificity), the overall capabilities of a number of analytical
procedures in combination may be investigated in order to ensure the quality of the
medicated feed.
Section 512(b) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. § 360b)
establishes the requirements for new animal drug approval. 21 C.F.R. § 514.1
specifies the information required to be submitted as part of the application and the
proper form for the submission. Section 514.1(b)(5)(vii) requires an applicant to
describe analytical procedures that should be capable of determining the active
component(s) within a reasonable degree of accuracy and of assuring the identity of
such components. Section 514.1(b)(5)(vii)(a) states that a description of practicable
1 This guidance has been prepared by the Office of New Animal Drug Evaluation in the Center for
Veterinary Medicine at the Food and Drug Administration.
This guidance represents the agency’s current thinking on this topic. It does not
create or confer any rights for or on any person and does not operate to bind
FDA or the public. You can use an alternative approach if the approach satisfies
the requirements of the applicable statute(s) and regulation(s). If you want to
discuss an alternative approach, contact the FDA staff responsible for
implementing this guidance. If you cannot identify the appropriate FDA staff,
call the appropriate number listed on the title page of this guidance.
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methods of analysis of adequate sensitivity to determine the amount of the new
animal drug in the final dosage form should be included.
FDA’s guidance documents, including this guidance, do not establish legally
enforceable responsibilities. Instead, guidances describe the Agency’s current
thinking on a topic and should be viewed only as recommendations, unless
specific regulatory or statutory requirements are cited. The use of the word
“should” in Agency guidances means that something is suggested or
recommended, but not required.
DISCUSSION
The objective of the analytical procedure should be clearly understood since this
will govern the validation characteristics that are evaluated. Typical validation
characteristics that may be considered are listed below:
- Specificity
- Linearity
- Range
- Accuracy
- Precision
- Limit of Detection
- Limit of Quantitation
- Robustness
Each of these validation characteristics is defined in the attached Glossary.
Approaches other than those set forth in this guidance may be acceptable. It is the
responsibility of the applicant to choose the validation procedure and protocol most
suitable for the product. However, it is important to remember that the main
objective of validation of an analytical procedure is to demonstrate that the
procedure is suitable for its intended purpose.
It is recommended that a well-characterized reference standard, with documented
purity, be used throughout the validation study. The degree of purity necessary
depends on the intended use.
For the sake of clarity, this document considers the various validation
characteristics in distinct sections. The arrangement of these sections reflects the
process by which an analytical procedure may be developed and evaluated.
In practice, it is recommended to design the experimental work such that the
appropriate validation characteristics can be considered simultaneously to provide a
sound, overall knowledge of the capabilities of the analytical procedure.
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Appropriate validation characteristics may include: specificity, linearity, range,
accuracy, and precision.
1. SPECIFICITY
It is recommended that an investigation of specificity be conducted during the
validation of the medicated feed assay. The procedures used to demonstrate
specificity will depend on the intended objective of the analytical procedure.
Identification of the analyte may be made by means of retention time of the
standard.
For chromatographic procedures, it is recommended that representative
chromatograms be used to demonstrate specificity, and individual feed components
and drug products be appropriately labeled. The chromatographic profile using
peak shape and tailing criteria may be used to indicate either co-eluting peaks or
sample matrix effects. The peak parameters should be in agreement between the
standard and analyte peaks. In addition, to ensure that the peaks are single
components, a diode array detector may be used to obtain peak purity information
for the analyte peaks in a variety of feed matrices. Similar considerations may be
given to other separation techniques.
For the assay, it is recommended that there be a demonstration of a lack of
interference by feed ingredients or other drug products that may be in the feed.
This may be done by demonstrating that the responses of a blank placebo made
from the feed ingredients and/or drug products, either separately or in combination,
are either different from the absorbance (for Ultraviolet (UV) methods) or retention
time (for Gas Chromatography (GC) and High Performance Liquid
Chromatography (HPLC) methods) of the analyte of interest or not significant (i.e.,
that the signal measured as a percent concentration is not greater than 10%). It is
recommended that additional information be provided showing that common feed
ingredients do not interfere with the detection system. If potential interference is
observed, it is recommended that the ingredient be evaluated by the complete
method. Some examples of interfering ingredients are clay agents (for flowability)
and pellet binding, molasses, grass meals (e.g., alfalfa), high mineral content, corn
cob meal, cottonseed by product meal, meat and bone meal, and fish meal. Many
methods developed to give good recovery in a simple corn-soy feed do not work
well when the analyte is added to high mineral feeds, and are not recommended.
Typically, 2-3 feed mixtures, based on the species that will be medicated,
geographical location where the feed may be prepared, and life cycle of the species
(e.g., starter, finisher), should be tested. For drug products, it is recommended that
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applicants consider the most common products that may typically be present within
the feed.
Applicable literature references demonstrating non-interference may be supplied in
lieu of actual testing.
2. LINEARITY
It is recommended that a linear relationship be evaluated across the range (see
section 3) of the analytical procedure. It may be demonstrated directly on the drug
substance by separate weighings (two separate weighings preferred) and/or dilution
of a standard stock solution, using the proposed procedure.
It is recommended that linearity be evaluated by visual inspection of a plot of
signals as a function of analyte concentration or content. If there is a linear
relationship, it is recommended that test results be evaluated by appropriate
statistical methods, for example, by calculation of a regression line by the method
of least squares. Data from the regression line itself may be helpful to provide
mathematical estimates of the degree of linearity. It is recommended that the
correlation coefficient (R) be at least 0.995. The regression line intercept should
not differ from zero if a single point calibration technique is used. This may be
demonstrated if the confidence limits of the intercept include zero or if the intercept
value is a small percentage of the target level. If the intercept is significantly
different from zero, then a single point calibration technique is not recommended.
For the establishment of linearity, a minimum of 5 concentrations, covering the
intended dosing range with one concentration 50% of the lowest dose, is
recommended. It is recommended that the sponsor contact CVM if other
approaches are used.
3. RANGE
The specified range should be derived from linearity studies and depends on the
intended application of the procedure. It may be established by confirming that the
analytical procedure provides an acceptable degree of linearity, accuracy, and
precision when applied to samples containing amounts of analyte within or at the
extremes of the specified range of the analytical procedure.
For the assay of a drug in a medicated feed, the range should be from 50 to 150
percent of the labeled concentration.
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4. ACCURACY
It is recommended that accuracy be established across the specified range of the
analytical procedure used for medicated feed assays.
It is recommended that two (2) typical feed matrices with known quantities of the
drug added be analyzed.
It is recommended that accuracy be assessed using a minimum of 15 - 20
determinations over the concentration levels covering the specified range for each
feed matrix tested (e.g. 3 - 4 concentrations (depending on the dose range) / 5
replicates each of the total analytical procedure). Recovery from fortified blank
matrix samples should be between 80 - 110%.
5. PRECISION
Validation of tests for assay of medicated feeds should include an investigation of
precision.
5.1. Repeatability
For fortified medicated feed samples, it is recommended that repeatability be
assessed using a minimum of 15 determinations covering the specified range for
the procedure (e.g., 3-4 concentrations / 5 replicates each).
For drugs incorporated into medicated feeds at greater than 10 ppm, the withinlaboratory
variation coefficient should be less than 5.0%. For drugs
incorporated into medicated feeds at less than 10 ppm, the within-laboratory
variation coefficient should be less than 7.5%.
5.2. Intermediate Precision
The extent to which intermediate precision should be established depends on the
circumstances under which the procedure is intended to be used. It is
recommended that the applicant establish the effects of random events on the
precision of the analytical procedure. It is recommended that variations to be
studied include days, analysts, equipment, etc. It is not recommended to study
these effects individually. Instead, the use of a statistical experimental design
(matrix) is encouraged (see Statistical Manual of the AOAC by W.J. Youden
and E.H. Steiner, 1975, page 33 for more information on statistical design of
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experiments). The performance of the method by a second independent
laboratory is encouraged.
5.3. Reproducibility
It is recommended that reproducibility be assessed by means of an interlaboratory
trial. It is recommended that reproducibility be considered in the
case of standardization of an analytical procedure.
5.4. Proof of Performance
It is recommended that proof of performance of the assay be demonstrated by
testing two (2) batches of the proposed medicated feed manufactured in mixing
equipment of the appropriate size and under conditions representative of typical
commercial processing. When feasible, batches should be manufactured using
different configurations of mixers.
It is recommended that a minimum of 10 determinations covering the specified
range for the procedure be made (e.g., 2 concentrations (high and low) / 5
replicates each). If the feed is pelletized, it is recommended that the mash and
the pelletized feed be tested separately. Results should be reported in both
concentration and percent label claim.
6. LIMIT OF DETECTION
There are several approaches for determining the limit of detection (LOD),
depending on whether the procedure is non-instrumental or instrumental.
Approaches other than those listed below may be used.
6.1. Based on Visual Evaluation
Visual evaluation may be used for non-instrumental or instrumental methods.
The detection limit may be determined by the analysis of samples with known
concentrations of analyte and by establishing the minimum level at which the
analyte can be reliably detected.
6.2. Based on Signal-to-Noise
It is recommended that this approach be applied only to analytical procedures
that exhibit baseline noise.
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Determination of the signal-to-noise ratio may be performed by comparing
measured signals from samples with known low concentrations of analyte with
those of blank samples, and establishing the minimum concentration at which
the analyte can be reliably detected. A signal-to-noise ratio of between 3 or 2:1
is generally recommended for estimating the detection limit.
6.3. Based on the Standard Deviation of the Response and the Slope
It is recommended that the LOD be expressed as:
LOD = 3.3 δ / S
where δ = the standard deviation of the responses and S = the slope of the
calibration curve. The slope S may be estimated from the calibration curve of
the analyte. The estimate of δ may be carried out in a variety of ways, for
example:
6.3.1. Based on the Standard Deviation of the Blank
It is recommended that the measurement of the magnitude of analytical
background response be performed by analyzing an appropriate number of
blank samples and calculating the standard deviation of these responses.
6.3.2. Based on the Calibration Curve
It is recommended that a specific calibration curve be studied using samples
containing an analyte in the range of the LOD. The residual standard deviation
of a regression line or the standard deviation of y-intercepts of regression lines
may be used as the standard deviation.
7. LIMIT OF QUANTITATION
Several approaches for determining the limit of quantitation (LOQ) are possible,
depending on whether the procedure is non-instrumental or instrumental.
Approaches other than those listed below may be used.
7.1. Based on Visual Evaluation
Visual evaluation may be used for non-instrumental or instrumental methods.
The LOQ may be determined by the analysis of samples with known
concentrations of analyte, and by establishing the minimum level at which the
analyte can be quantified with acceptable accuracy and precision.
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7.2. Based on Signal-to-Noise Approach
It is recommended that this approach be applied only to analytical procedures
that exhibit baseline noise. Determination of the signal-to-noise ratio may be
performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples, and establishing the
minimum concentration at which the analyte can be reliably quantified. A
signal-to-noise ratio of 10:1 is recommended.
7.3. Based on the Standard Deviation of the Response and the Slope
The LOQ may be expressed as:
LOQ = 10 δ / S
where δ = the standard deviation of the responses and S = the slope of the
calibration curve. The slope S may be estimated from the calibration curve of
the analyte. The estimate of δ may be carried out in a variety of ways, for
example:
7.3.1. Based on the Standard Deviation of the Blank
Measurement of the magnitude of analytical background response may be
performed by analyzing an appropriate number of blank samples and calculating
the standard deviation of these responses.
7.3.2. Based on the Calibration Curve
It is recommended that a specific calibration curve be studied using samples
containing an analyte in the range of the LOQ. The residual standard deviation
of a regression line or the standard deviation of y-intercepts of regression lines
may be used as the standard deviation.
8. ROBUSTNESS / RUGGEDNESS
It is recommended that the evaluation of robustness be considered during the
development phase and demonstrated during the analytical validation phase.
Robustness depends on the type of procedure under study. It should show the
reliability of an analysis with respect to deliberate variations in method parameters.
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If measurements are susceptible to variations in analytical conditions, it is
recommended that the analytical conditions be suitably controlled or a
precautionary statement be included in the procedure. One consequence of the
evaluation of robustness should be that a series of system suitability parameters
(e.g., resolution test) are established to ensure that the validity of the analytical
procedure is maintained whenever used.
Examples of typical variations are:
-stability of analytical solutions and feed extracts; and
-extraction time.
(Note: it is recommended that results of the stability studies of the
analytical solutions and feed extracts be included in the procedure)
In the case of high performance liquid chromatography (HPLC), examples of
typical variations are:
-influence of variations of pH in a mobile phase;
-influence of variations in mobile phase composition;
-different columns (different lots and/or suppliers); and
-temperature-flow rate.
In the case of gas chromatography (GC), examples of typical variations are:
-different columns (different lots and/or suppliers); and
-temperature-flow rate.
9. SYSTEM SUITABILITY TESTING
System suitability testing is an integral part of many analytical procedures. The
tests are based on the concept that the equipment, electronics, analytical operations,
and samples to be analyzed constitute an integral system that can be evaluated as
such. System suitability test parameters to be established for a particular procedure
depend on the type of procedure being validated and may include, for example, data
acceptability testing for feed controls. It is recommended that system suitability
tests and criteria for the HPLC or GC detection system be evaluated. Performance
specifications for critical reagents and steps, such as solid phase extraction, should
be included when appropriate. If specific tests and criteria are used, then the
recommended actions taken if performance does not meet the criteria should be
determined. Additional information is available in Pharmacopoeias.
10. RECOMMENDED DATA
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It is recommended that data collected during validation and formulae used for
calculating validation characteristics be submitted for each feed type and discussed
as outlined below:
Specificity:
It is recommended that representative sample sets of chromatograms be
provided so that recalculation can be performed, including:
Baseline / mobile phase
Extraction solvent
Feed ingredient placebo that cause interference
Other drug product placebo that causes interference
Standards
Samples (high and low concentration, different feed mixtures)
Retention times and a comparison of relative retention times should be
provided.
Tabular listing of feed mixture ingredients and other drug products tested
should be provided.
Linearity & Range:
It is recommended that the correlation coefficient, y-intercept, slope of the
regression line, and residual sum of squares be submitted. A plot of the data
should be included. In addition, an analysis of the deviation of the actual data
points from the regression line may also be helpful for evaluating linearity.
Accuracy:
It is recommended that accuracy be reported as percent recovery by the assay of
known added amount of analyte in the sample or as the difference between the
mean and the accepted true value together with the confidence intervals.
Tabular listing of feed mixture ingredients used should be provided.
For each feed matrix studied, it is recommended that the complete set of data
including weighings, sample and standard preparation, chromatography,
calculations, and results be provided. A representative set of chromatograms
should be provided and, for the concentration(s) in between, a table of relevant
parameters should be provided. All individual area or height measurements for
controls, standards, and samples and all other information such as sample
weights, standard concentrations, and dilutions should also be provided.
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Precision:
It is recommended that the standard deviation, relative standard deviation
(coefficient of variation), and confidence interval be reported for each type of
precision investigated.
It is recommended that the complete set of data including weighings, sample
and standard preparation, chromatography, calculations, and results be
provided. A representative set of data should be provided and, for the
concentration(s) in between, a table of relevant parameters should be provided
for each type of precision investigated.
Limit of Detection:
It is recommended that the limit of detection and the method used for
determining the detection limit be presented. If the LOD is determined based
on visual evaluation or based on signal-to-noise ratio, the presentation of the
relevant chromatograms may be considered acceptable for justification.
In cases where an estimated value for the LOD is obtained by calculation or
extrapolation, this estimate may subsequently be validated by the independent
analysis of a suitable number of samples known to be near, or prepared at, the
LOD.
Limit of Quantitation:
It is recommended that the limit of quantitation and the method used for
determining the LOQ be presented. The limit should be subsequently
confirmed by the analysis of a suitable number of samples known to be near, or
prepared at, the LOQ.
Robustness/Ruggedness:
It is recommended that tabular representation including conditions tested,
retention times, tailing factors, effects on resolution, and potency be presented.
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GLOSSARY
1. ANALYTICAL PROCEDURE
The analytical procedure refers to the way an analysis is performed. It describes in
detail the steps that should be followed to perform each analytical test. This may
include, but is not limited to, the sample, the reference standard and the reagents
preparations, use of the apparatus, generation of the calibration curve, and use of
the formulae for the calculation.
2. SPECIFICITY
Specificity is the ability to assess unequivocally the analyte in the presence of
components that may be expected to be present. Typically, these might include
impurities, degradation products, matrix, other approved drugs, etc.
Lack of specificity of an individual analytical procedure may be compensated for
by other supporting analytical procedure(s).
This definition includes the following:
Identification: to ensure the identity of an analyte.
Assay (content or potency): to provide an exact result which allows an
accurate statement on the content or potency of the analyte in a sample.
3. LINEARITY
The linearity of an analytical procedure is its ability (within a given range) to obtain
test results that are directly proportional to the concentration (amount) of analyte in
the sample.
4. RANGE
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations)
for which it has been demonstrated that the analytical procedure has a suitable level
of precision, accuracy, and linearity.
5. ACCURACY
The accuracy of an analytical procedure refers to the closeness of agreement
between the value that is accepted either as a conventional true value or an accepted
reference value, and the value found.
This is sometimes termed trueness.
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6. PRECISION
The precision of an analytical procedure expresses the closeness of agreement
(degree of scatter) between a series of measurements obtained from multiple
sampling of the same homogenous sample under the prescribed conditions.
Precision may be considered at three levels: repeatability, intermediate precision,
and reproducibility.
Precision is investigated using homogenous, authentic samples. However, if it is
not possible to obtain a homogenous sample, it may be investigated using
artificially prepared samples or a sample solution (although extraction variability
will not be measured).
The precision of an analytical procedure is usually expressed as the variance,
standard deviation, or coefficient of variation of a series of measurements.
6.1. Repeatability: Repeatability expresses the precision under the same
operating conditions over a short interval of time. Repeatability is also termed
intra-assay precision.
6.2. Intermediate precision: Intermediate precision expresses within-laboratories
variations: different days, different analysts, different equipment, etc.
6.3. Reproducibility: Reproducibility expresses the precision between
laboratories (collaborative or transfer studies, usually applied to standardization
of methodology).
7. LIMIT OF DETECTION
The limit of detection of an individual analytical procedure is the lowest amount of
analyte in a sample that can be detected, but not necessarily quantitated as an exact
value.
8. LIMIT OF QUANTITATION
The limit of quantitation of an individual analytical procedure is the lowest amount
of analyte in a sample that can be quantitatively determined with suitable precision
and accuracy. The quantitation limit is a parameter of quantitative assays for low
levels of compounds in sample matrices and is used particularly for the
determination of impurities and/or degradation products.
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9. ROBUSTNESS / RUGGEDNESS
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters. Robustness
provides an indication of its reliability during normal usage.
10. SYSTEM SUITABILITY
A procedure run prior to the individual analytical analysis to demonstrate that the
instrument, column, mobile phase, etc., parameters are within defined criteria.
Adequate system suitability is demonstrated before proceeding with the analysis.